We have completed a genome-wide screen for chromosomal loci of susceptibility genes that control hyperoxia-induced pulmonary injury in an intercross (B6C3F2) cohort derived from susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) mice. Significant and suggestive quantitative trait loci (QTLs) were identified on chromosomes 2 and 3, respectively. A candidate gene within the chromosome 2 QTL is Nfe2l2 (nuclear factor, erythroid derived 2, like 2), which encodes a transcription factor NRF2 (NF-E2 related factor 2, Nrf2). NRF2 has been identified as an antioxidant response element (ARE)-mediated positive regulator of detoxifying enzymes genes for protecting cells against electrophile toxicity, oxidative stress, and carcinogenicity. To test the hypothesis that Nrf2 is a candidate gene for differential susceptibility in B6 and C3 mice, we sequenced Nrf2 in both strains. Analysis of the Nrf2 promoter in B6 and C3 mice revealed a potentially important variation between them. B6 mice possess a T to C substitution at -336, which is predicted to add a Sp1 transcription factor-binding site in B6 mice compared to C3. The polymorphism segregated with hyperoxia susceptibility in the B6C3F2 cohort, suggesting that the polymorphism is potentially an important determinant of suceptibility to hyperoxic lung injury in this model. We tested the hypothesis that NRF2 contributes to pulmonary protection against hyperoxic injury by exposing mice with site-directed mutation of Nrf2 (Nrf2-/-) and wild type (Nrf2+/+) mice to hyperoxia. Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury were significantly greater in Nrf2-/- mice compared to Nrf2+/+ mice after exposure to hyperoxia. Consistent with a protective role for Nrf2 in hyperoxic lung injury, mRNA expression of multiple antioxidant and phase 2 genes, as well as antioxidant enzyme activities, were significantly lower in Nrf2-/- mice compared to Nrf2+/+ mice after hyperoxia. These studies have identified Nrf2 as an important susceptibility gene in the pathogenesis of oxidant-induced lung pathology in the mouse, and may have important implications for understanding similar processes in susceptible human populations.